Elsevier

Journal of Hepatology

Volume 60, Issue 2, February 2014, Pages 298-305
Journal of Hepatology

Research Article
A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury

https://doi.org/10.1016/j.jhep.2013.09.013Get rights and content

Background & Aims

Hepatic stellate cells (HSCs) that express glial fibrillary acidic protein (GFAP) are located between the sinusoidal endothelial cells and hepatocytes. HSCs are activated during liver injury and cause hepatic fibrosis by producing excessive extracellular matrix. HSCs also produce many growth factors, chemokines and cytokines, and thus may play an important role in acute liver injury. However, this function has not been clarified due to unavailability of a model, in which HSCs are depleted from the normal liver.

Methods

We treated mice expressing HSV-thymidine kinase under the GFAP promoter (GFAP-Tg) with 3 consecutive (3 days apart) CCl4 (0.16 μl/g; ip) injections to stimulate HSCs to enter the cell cycle and proliferate. This was followed by 10-day ganciclovir (40 μg/g/day; ip) treatment, which is expected to eliminate actively proliferating HSCs. Mice were then subjected to hepatic ischemia/reperfusion (I/R) or endotoxin treatment.

Results

CCl4/ganciclovir treatment caused depletion of the majority of HSCs (about 64–72%), while the liver recovered from the initial CCl4-induced injury (confirmed by histology, serum ALT and neutrophil infiltration). The magnitude of hepatic injury due to I/R or endotoxemia (determined by histopathology and serum ALT) was lower in HSC-depleted mice. Their hepatic expression of TNF-α, neutrophil chemoattractant CXCL1 and endothelin-A receptor also was significantly lower than the control mice.

Conclusions

HSCs play an important role both in I/R- and endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects.

Introduction

The perisinusoidal hepatic stellate cells (HSCs) constitute 8–12% of the liver cell population, express glial fibrillary acidic protein (GFAP) and/or desmin, and are the major storage site of retinoids [1]. During liver injury, HSCs undergo activation characterized by the loss of retinoids, expression of α-smooth muscle actin and differentiation into proliferating myofibroblast-like cells. Activated HSCs produce excessive extracellular matrix (ECM) and exhibit increased expression of tissue inhibitors of metalloproteinases and reduced or unchanged expression of matrix metalloproteinases [2], [3], [4], thus becoming the major cell type responsible for hepatic fibrosis [5]. Activated HSCs are postulated to contribute to portal hypertension by their high contractility and up-regulation of the powerful vasoconstrictor endothelin-1 (ET-1) and its receptors [6], [7].

HSCs express intercellular adhesion molecule-1 [8], produce various cytokines and chemokines [8], [9], [10], and thus can play an important role in hepatic inflammation. Gram-negative bacterial endotoxin (lipopolysaccharide: LPS) stimulates the synthesis of nitric oxide (NO), ET-1, tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both quiescent and activated HSCs; LPS-challenged HSCs stimulate NO synthesis, inhibit DNA synthesis and cause apoptosis of cultured hepatocytes [11], [12], [13], [14].

Recent work demonstrates that HSCs also influence hepatic immunological functions. HSCs induce apoptosis of allogeneic CD4+ and CD8+ T cells [8], [15], present bacterial lipid antigens to NKT cells [16], expand immunosuppressive regulatory T cells [8], and render dendritic cells immunosuppressive [10]. Furthermore, HSCs secrete powerful antioxidant protein(s) that protect hepatocytes from ischemia/reperfusion (I/R) injury [17].

Thus, HSCs are highly versatile cells that can profoundly influence hepatic structure and functions in physiology and pathology. Most of the in vivo work confirming their role in hepatic pathology has been focused on fibrosis. A fungal metabolite gliotoxin was found to cause apoptosis of activated rat and human HSCs in vitro, and of rat HSCs in vivo resulting in resolution of fibrosis [18], [19]. However, gliotoxin also induces apoptosis of KCs and endothelial cells in the fibrotic liver [20], [21]. Ebrahimkhani et al. [22] administered gliotoxin into bile duct-ligated mice in conjugation with the single-chain antibody C1–3, which recognizes synaptophysin expressed by activated HSCs [23]; C1–3-gliotoxin caused resolution of fibrosis by selectively depleting HSCs. It was recently reported, using a similar mouse model described in the present study that concomitant treatment of B6.Cg-Tg(Gfap-Tk)7.1Mvs/J transgenic mice with ganciclovir promoted depletion of HSCs, and caused amelioration of CCl4-induced fibrosis and hepatic injury [24]. However, the role of HSCs in acute injury to the normal liver has not yet been evaluated. Here, we show amelioration of I/R- and endotoxin-induced acute injury to otherwise normal HSC-depleted liver, suggesting HSCs’ critical role in pathologies unrelated to activation-dependence.

Section snippets

Animals

The protocols were approved by the IACUC according to NIH guidelines. Wild-type male C57BL/6 (WT-B6) and B6.Cg-Tg(Gfap-Tk)7.1Mvs/J (GFAP-Tg) mice were from The Jackson laboratory. GFAP-Tg mice express the herpes simplex virus thymidine kinase (HSV-TK) transgene under the GFAP promoter [25]. HSV-TK phosphorylates non-toxic ganciclovir (GCV) to GCV-monophosphate, which is converted to GCV-triphosphate by cellular guanylate kinase; phosphorylated GCV incorporates into the DNA causing death of

Characterization of the CCl4 effect on WT mice

To ensure that at the end of GCV treatment of mice, there is no residual hepatic injury due to earlier CCl4 administration before subjecting them to I/R or endotoxemia, WT-B6 mice were sacrificed a day after the third CCl4 injection or after 10 days of GCV treatment following termination of CCl4. There was significant hepatic injury the day after third CCl4 administration (Fig. 1A and B, middle panels), in both centrilobular and periportal areas (Supplementary Fig. 2), accompanied by

Discussion

The role of activated HSCs in hepatic fibrosis and cirrhosis has been established [5], [33]. Recent research with cell culture studies and some indirect in vivo evidence indicate that quiescent or transiently activated HSCs (found in the liver during early phase of acute injury) can significantly influence metabolic and hemodynamic properties of the liver [7]. However, the lack of animal models in which HSCs are selectively depleted has been a major drawback to demonstrate unequivocally their

Financial support

NIH PO1A1081678 and VA Merit Review Award 1IO1BX001174, and Department of Surgery, University of Cincinnati and Children’s Hospital Medical Center, Cincinnati.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The underlying research reported in the study was funded by the NIH Institutes of Health.

Acknowledgement

We thank Ms. Rebecca B. Schuster for excellent technical assistance.

References (57)

  • C. Fondevila et al.

    Hepatic ischemia/reperfusion injury – a fresh look

    Exp Mol Pathol

    (2003)
  • N. Kawada et al.

    ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1

    Biochem Biophys Res Commun

    (1999)
  • R.Z. Zhao et al.

    TNF-alpha induces interleukin-8 and endothelin-1 expression in human endothelial cells with different redox pathways

    Biochem Biophys Res Commun

    (2005)
  • J.C. Cutrn et al.

    Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning

    Free Radic Biol Med

    (2002)
  • M.J. Edwards et al.

    The involvement of kupffer cells in carbon tetrachloride toxicity

    Toxicol Appl Pharmacol

    (1993)
  • A. Geerts

    History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells

    Semin Liver Dis

    (2001)
  • J.P. Iredale

    Hepatic stellate cell behavior during resolution of liver injury

    Semin Liver Dis

    (2001)
  • N.C. Henderson et al.

    Liver fibrosis: cellular mechanisms of progression and resolution

    Clin Sci

    (2007)
  • D.C. Rockey

    Hepatic blood flow regulation by stellate cells in normal and injured liver

    Semin Liver Dis

    (2001)
  • C.R. Gandhi

    Hepatic stellate cells

  • A. Dangi et al.

    Selective expansion of allogeneic regulatory T cells by hepatic stellate cells: role of endotoxin and implications for allograft tolerance

    J Immunol

    (2012)
  • M. Pinzani et al.

    Cytokine receptors and signaling in hepatic stellate cells

    Semin Liver Dis

    (2001)
  • T.L. Sumpter et al.

    Hepatic stellate cells undermine the allostimulatory function of liver myeloid dendritic cells via STAT3-dependent induction of IDO

    J Immunol

    (2012)
  • C.R. Gandhi et al.

    Endotoxin causes up-regulation of endothelin receptors in cultured hepatic stellate cells via nitric oxide-dependent and -independent mechanisms

    Br J Pharmacol

    (2000)
  • T. Uemura et al.

    Inhibition of DNA synthesis in cultured hepatocytes by endotoxin-conditioned medium of activated stellate cells is transforming growth factor-β- and nitric oxide-independent

    Br J Pharmacol

    (2001)
  • C. Thirunavukkarasu et al.

    Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes

    J Cell Physiol

    (2005)
  • C. Thirunavukkarasu et al.

    Mechanisms of endotoxin-induced nitric oxide, interleukin-6 and tumor necrosis factor-α production in activated rat hepatic stellate cells: role of p38MAPK

    Hepatology

    (2006)
  • M.C. Yu et al.

    Inhibition of T cell responses by hepatic stellate cells via B7–H1 mediated T cell apoptosis

    Hepatology

    (2004)
  • Cited by (93)

    • Endotoxin-Stimulated Hepatic Stellate Cells Augment Acetaminophen-Induced Hepatocyte Injury

      2022, American Journal of Pathology
      Citation Excerpt :

      Interestingly, however, IFN-β administration did not restore APAP-induced injury in HSC-depleted mice to levels similar to LPS/APAP-treated HSC-sufficient mice, suggesting that the process involves additional mechanisms. However, from the previous work showing protection from LPS- or ischemia-reperfusion–induced injury in HSC-depleted mice24 and the results of the present study, it is apparent that in the absence of HSCs, Kupffer cells are unable to produce significant levels of injurious mediators. Data from the current study indicate that nuclear IRF1 translocation can be an important mechanism of LPS/APAP-induced liver damage.

    View all citing articles on Scopus

    These authors contributed equally to this investigation.

    View full text